The presence and quantity of specific RNA polymerase (Pol) II transcripts in biospecimens, that reflect gene expression patterns, are increasingly being used as biomarkers to make major patient care decisions (e.g., MammaPrintTM, array assay for 70 mRNAs). However, mRNA molecules in biospecimens are highly susceptible to degradation by RNases during sample collection, handling, and storage. Reliable data obtained by transcriptome analysis of biospecimens, with gene arrays or RNA-Sequencing, is highly dependent on the quality of the RNA analyzed. Current standard measures of RNA integrity focus on 28S and 18S ribosomal RNA. However, diagnostic and prognostic gene expression markers of cancer focus on changes in mRNAs which are RNA Pol II transcripts. The 5'ends of Pol II RNAs are unique in that they have a 5'm7GpppN cap. We will use new technologies to identify a panel of sentinel Pol II RNA transcripts in human colon, breast and liver biospecimens that mirror mRNA quality and use these RNAs to develop a 3'/5'PCR based assay that more accurately assesses the integrity of Pol II transcripts in biospecimens. We developed a new approach to identify and characterize all Pol II transcripts present in biospecimens by isolating 5'm7G capped RNAs and analyzing them with RNA-sequencing technologies (Illumina). This allows us to identify and quantitate both protein coding and non-coding regulatory RNAs in biospecimens. It also allows us to define the entire length of each RNA, define their 5'-3'pattern of degradation, and develop a qRT-PCR based assay of their 3'and 5'regions to measure their level of intactness. The degradation patterns of Pol II RNA transcripts will be assessed in freshly collected human colon, breast and liver biospecimens (normal and cancer) after increasing times at room temperature and commonly used handling procedures. This analysis will identify a panel of candidate sentinel RNAs that can be used to develop an assay for mRNA integrity in biospecimens. The Specific Aims are: 1) To identify candidate sentinel Pol II RNAs that mirror mRNA decay in specific biospecimens (see Example) and;2) To develop a 5'/3'quantitative reverse transcription PCR (qRT-PCR) assay that measures the intactness of sentinel RNA transcripts in specific biospecimens to better measure mRNA integrity. This study stimulates technology innovation by combining a new RNA purification technology for RNA Pol II transcripts and next generation RNA-sequencing to identify sentinel RNAs and developing a practical assay for RNA integrity in biospecimens. Our studies will optimize the use of both currently stored and future collections of biospecimens in identifying gene expression biomarkers for the early diagnosis, prognosis, and response to therapy of cancer patients. The long-term goal of this technology is improving patient care and therapeutic outcomes by better determining the quality of RNA in biospecimens before conducting diagnostic or predictive gene expression tests. Two international experts in colon (Dr. Burt) and breast (Dr. Buys) cancer will provide expert advice during the development of this assay. PUBLIC HEALTH RELEVANCE: Thousands of clinical biospecimens are collected every year at cancer centers throughout the United States for the proper diagnosis and prognosis of breast, colon, and liver cancer. RNA molecules in these biospecimens are increasingly being used in diagnostic and prognostic testing and more recently in major therapeutic decisions. Unfortunately, the procedures for collection and storage of these biospecimens vary and many times not optimized to ensure sample integrity and quality. The purpose of this study is to develop an innovative technology to identify and assay a subset of mRNA molecules that are highly susceptible to degradation and can be used as molecular "sentinels" to better determine the quality of mRNA in clinical biospecimens before measuring panels of gene expression biomarkers. The long term goal of this study is improving patient care and therapeutic outcomes by better determining the quality of biospecimens before running multi gene expression diagnostic tests.